Scodaphoresis and methods and apparatus for moving and concentrating particles

ABSTRACT

Methods and apparatus for moving and concentrating particles by applying an alternating driving field and an alternating field that alters mobility of the particles. The driving field and mobility-varying field are correlated with one another. The methods and apparatus may be used to concentrate DNA or RNA in a medium, for example. Methods and apparatus for extracting particles from one medium into another involve applying an alternating driving field that causes net drift of the particles from the first medium into the second medium but no net drift of the particles in the second medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/360,640, filed Jan. 27, 2012 which is a continuation of U.S.application Ser. No. 10/597,307, which is a 371 of InternationalApplication No. PCT/CA2005/000124 filed on 2 Feb. 2005, which claims thebenefit of the filing date of U.S. application No. 60/540,352 filed on 2Feb. 2004 and U.S. application No. 60/634,604 filed on 10 Dec. 2004,which are all hereby incorporated herein by reference.

TECHNICAL FIELD

The invention relates to methods and apparatus for moving andconcentrating particles. The invention has application, for example, inmoving and/or concentrating particles of a wide range of types. Someexamples of particles that can be moved and/or concentrated byembodiments of the invention include molecules such as nucleic acids,proteins, other bio-macromolecules, inorganic or inorganic ions, andother particles of molecular size and larger including suitable magneticparticles, and other particles.

BACKGROUND

Pathogens and certain diseases can be identified in the environment orin a patient by detecting DNA associated with the pathogen or disease inenvironmental samples, body fluids, water, or other contaminatedsolutions. DNA can also be extracted from crime scenes and associatedevidence. It is generally necessary to concentrate DNA before the DNAcan be identified. DNA can be concentrated by filtration. However,filtration technologies are inefficient. Filters fine enough to trapDNA, viruses or the like are easily clogged with other debris. There isa general need for technologies capable of concentrating DNA and similarmaterials and/or extracting relatively pure DNA from contaminatedsolutions.

Laborious and/or expensive purification methods are often employed toprepare samples containing nucleic acids for biochemical assays. Thepolymerase chain reaction (PCR) can be used to amplify theconcentrations of nucleic acids such as DNA and RNA. However, PCR can beundesirably expensive, especially for large volume samples.

Electrophoresis involves directing the movement of charged particles ina medium, such as a gel or liquid solution by applying an electric fieldacross the medium. The electric field may be generated by applying apotential across electrodes that are placed in contact with the mediumsuch that electric current can be conducted into the medium. Themovement of the particles in the medium is affected by the magnitude anddirection of the electric field, the electrophoretic mobility of theparticles and the mechanical properties, such as viscosity, of themedium. Through electrophoresis, particles that are distributed in amedium can be transported through the medium. Electrophoresis iscommonly used to transport nucleic acids (such as DNA or RNA) throughgel substrates. Since different species have different electrophoreticmobilities, electrophoresis may be used to separate different speciesfrom one another. Conventional electrophoresis techniques are largelylimited in application to the linear separation of charged particles.Using conventional electrophoresis techniques, a direct current (DC)electric field or an alternating pulsed-field electrophoretic (PFGE)field is typically applied to a medium so that particles in the mediumare transported toward an electrode.

Electrophoresis may be used to transport fragments of DNA or othermicroscopic electrically charged particles. Various electrophoresismethods are described in Slater, G. W. et al. Electrophoresis 2000, 21,3873-3887. Electrophoretic particle transport is typically performed inone dimension by applying a direct current (DC) electric field betweenelectrodes on either side of a suitable electrophoresis gel. Theelectric field causes electrically charged particles in the gel to movetoward one of the electrodes. Electrophoresis is typically used toseparate particles of different types from one another.

Electrophoresis can also be used to concentrate particles in aparticular location. A problem that can interfere with the successfuluse of electrophoresis for concentrating particles in some applicationsis that there must be an electrode at the location where the particlesare to be concentrated. Electrochemical interactions between theelectrodes and particles can degrade certain kinds of the particles. Forexample, where the particles comprise DNA, the DNA can be damaged byelectrochemical interactions at the electrodes.

Electric fields present during conventional direct currentelectrophoresis are divergence-free everywhere except at electrodeswhich can source or sink electric current. Electrophoresis is typicallyapplied in cases where particles are caused to move toward an electrode.

An asymmetric alternating current (AC) waveform can cause net drift ofelectrophoretic particles due to nonlinearity of the relationshipbetween particle speed and applied electric field. This effect can beused to cause particles to move in one dimension as described inChacron, M. J., et al. Phys. Rev. E 1997, 56, 3446-3450; Frumin, L. L,et al. Phys. Chem. Commun. 2000, 11; and, Frumin, L. L. et al. Phys.Rev. E 2001, 64, 021902.

Pohl, H. A., Dielectrophoresis: The Behavior of Neutral Matter inNonuniform Electric Fields Cambridge University Press, Cambridge, UK1978; Asbury, C. L., et al., Electrophoresis 2002, 23, 2658-2666; andAsbury, C. L., et al. Biophys. J. 1998, 74, 1024-1030 disclose thatdielectrophoresis can be applied to concentrate DNA in two or moredimensions. However, practical applications of dielectrophoresis requireundesirably high electric field gradients.

One can isolate particles which have been separated from other particlesby electrophoresis by cutting out the portion of the medium in which theparticles have been carried by electrophoresis. The particles can beseparated from the medium by using various purification techniques.

References which describe methods for DNA separation include: Slater etal. The theory of DNA separation by capillary electrophoresis CurrentOpinion in biotechnology 2003 14:58-64; Slater et al. U.S. Pat. No.6,146,511 issued 14 Nov. 2000; Frunin et al. Nonlinear focusing of DNAmacromolecules Phys. Rev. E 64:021902; Griess et al. Cyclic capillaryelectrophoresis Electrophoresis 2002, 23,2610-2617 Wiley-VCH Verlag GmbH& Co. Weinheim (2002). References which describe the use of fields toseparate particles include: Bader et al. U.S. Pat. No. 5,938,904 issuedon Aug. 17, 1999; Bader et al. U.S. Pat. No. 6,193,866 issued on 27Feb., 2001; Tessier et al: Strategies for the separation ofpolyelectrolytes based on non-linear dynamics and entropic ratchets in asimple microfluidic device Appl. Phys. A 75, 285-291 (2002); Chacron etal. Particle trapping and self-focusing in temporally asymmetricratchets with strong field gradients Phys. Rev. B 56:3 3446-3550(September 1997); Dean et al. Fluctuation driven ratchets: molecularmotors Phys. Rev. Lett. 72:11 1766-1769 (14 Mar. 1994); Bier et al.Biasing Brownian motion in different directions in a 3-state fluctuatingpotential and an application for the separation of small particles Phys.Rev. Lett. 76:22 4277-4280 (27 May 1996); Magnasco, forced thermalratchets Phys. Rev. Lett. 71:10 1477-1481 (6 Sep. 1993).

There remains a need for methods for moving and/or concentratingparticles that improve on prior art methods and avoid limitations ofprior art methods in specific applications. There also remains a needfor effective methods for extracting materials such as DNA from mediasuch as gels.

SUMMARY OF THE INVENTION

One aspect of the invention provides methods for causing motion ofparticles in a medium. The methods may be used for concentratingparticles and/or for separating particles of different types from oneanother. Such methods comprise applying a time-varying driving field tothe particles. The driving field applies a time-varying driving forcealternating in direction to the particles. The methods also compriseapplying a mobility-varying field to the particles. The mobility-varyingfield is one or both of: different in type from the driving field, andnon-aligned with the driving field. The driving field andmobility-varying field are applied simultaneously during a period andthe mobility-varying field causes a mobility of the particles in themedium to be time dependent during the period, in a manner having anon-zero correlation with the driving field over the period. Thesemethods may be called SCODA methods.

Another aspect of the invention provides methods and apparatus forextracting charged particles from a medium. These methods may be appliedto extracting particles from a medium which have been concentrated bySCODA and may also be applied to extracting from a medium particleswhich have not been concentrated by SCODA. A buffer in an extractionreservoir is placed to abut a medium containing the particles to beextracted at a buffer-gel interface. Electrodes are provided on eachside of the buffer-gel interface. By applying a pulsed voltage potentialto the electrodes (wherein the time-averaged electric field is zero),zero-integrated-field electrophoresis (ZIFE) is applied to thebuffer-gel interface to direct the particles in the gel into theextraction reservoir, where the particles are collected andconcentrated.

A method according to one aspect of the invention comprises placing abuffer extraction reservoir next to a gel solution containing thecharged particles to be extracted; applying ZIFE to the buffer-gelinterface to direct the particles into the extraction reservoir; andcollecting and concentrating the particles in the extraction reservoir.A pipette or other device may then be used to suction the particles fromthe extraction reservoir.

In some embodiments of the invention, the apparatus comprises a gel boatholding a gel that contains the charged particles to be extracted. Acapillary containing a small amount of buffer is inserted into the gelsolution. A pipette or other device is provided in the capillary forsuctioning the particles that have collected in the buffer. Electrodesare provided on each side of the buffer-gel interface for generating anelectric field.

Further aspects of the invention and features of specific embodiments ofthe invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate non-limiting embodiments of the invention,

FIGS. 1A through 1I are examples of possible waveforms for driving andmobility-modifying fields;

FIG. 2 is a plot showing a numerical simulation of the path of aparticle;

FIGS. 3, 3A, 3B, 3C and 3D are schematic diagrams of apparatus that maybe used to practice embodiments of the invention;

FIG. 4 is an example plot of measured DNA velocity as a function ofapplied electric field;

FIG. 4A is a plot illustrating time averaged particle velocity in anapparatus like that of FIG. 3 as a function of radial distance from theorigin;

FIG. 4B is a plot showing the measured DNA spot distance from the originas a function of time;

FIG. 5 shows estimated particle velocity as a function of electric fieldstrength for three molecules in a sieving matrix comprising covalentlybound oligonucleotides;

FIG. 6 is a schematic diagram of apparatus that may be used to explorescodaphoresis using an electric driving field and a thermalmobility-varying field;

FIG. 7 is a schematic diagram of apparatus that may be used to explorescodaphoresis using an electric driving field and an opticalmobility-varying field;

FIG. 7A shows waveforms from the apparatus of FIG. 7;

FIGS. 8A, 8B, 8C and 8D show optical mask patterns that may be used tocause concentration of particles at an array of spots;

FIG. 8E shows an array of spots at which particles can be concentratedusing the masks of FIGS. 8A through 8D;

FIG. 9 is a schematic view of apparatus for producing cyclic variationsin viscosity of a medium;

FIGS. 10A, 10B, 10C and 10D show schematically apparatus for usingmagnetic fields to alter the mobility of particles;

FIG. 11 is a graphical illustration of an exemplary electric field pulseused in ZIFE;

FIG. 12A is a cross-sectional elevation view of an extraction apparatusin accordance with a particular embodiment of the present invention,illustrating molecules of DNA in a solution prior to extraction;

FIG. 12B is a cross-sectional elevation view of the apparatus of FIG.12A, illustrating molecules of DNA extracted from a solution andconcentrated in a small amount of buffer;

FIG. 12C is a plan view of an extraction apparatus similar to that shownin FIG. 12A;

FIG. 13 shows a glass capillary in an extraction experiment using theapparatus and method in accordance with a particular embodiment of theinvention;

FIG. 14 is a graph illustrating the DNA fragment velocity during anexperiment as a function of fragment length and cycle times; and,

FIG. 15 shows a comparison between a DNA fragment mix and the fragmentdistribution of the same mix, after extraction.

DESCRIPTION

Throughout the following description, specific details are set forth inorder to provide a more thorough understanding of the invention.However, the invention may be practised without these particulars. Inother instances, well known elements have not been shown or described indetail to avoid unnecessarily obscuring the invention. Accordingly, thespecification and drawings are to be regarded in an illustrative, ratherthan a restrictive, sense.

Scodaphoresis (which is referred to herein by the coined acronym SCODA)describes methods for moving and/or concentrating particles in a medium.SCODA is an acronym for synchronous coefficient of drag alteration.“phoresis” is a combining form meaning to carry or transmit.Scodaphoresis involves exposing particles that are to be moved and/orconcentrated to two time-varying fields or stimuli. A first one of thefields results in a force f(t) that drives motion of the particles inthe medium. The direction of particle motion caused by the interactionof the particle with the first field varies in time. The first field mayprovide a driving force that averages to zero over an integral number ofcycles of the first field.

A second one of the fields alters the mobility of the particles in themedium according to a function g(t). The first and second fields aresuch that f(t) and g(t) have a non-zero correlation over a time periodof interest. Achieving such a non-zero correlation can be achieved invarious ways. In some embodiments, f(t) and g(t) are each time varyingat the same frequency and f(t) and g(t) are synchronized so that thereis a substantially constant phase relationship between f(t) and g(t). Inother embodiments, f(t) has a frequency that is twice that of g(t).

Application of the fields to the particles causes a net drift of theparticles. This net drift can be harnessed to separate particles ofdifferent types or to concentrate particles in selected areas, or both.As discussed below, the first and second fields may be of the same type(homogeneous SCODA) or of different types (heterogeneous SCODA).

As a demonstration of SCODA, consider the case where:f(t)=sin(ωt), g(t)=sin(ωt), and v(f(t), g(t))=f(t)×(μ₀+μ₁ g(t))  (1)where μ₀ is the unperturbed mobility of the particle in the medium andμ₁ is the susceptibility of the mobility to g(t). It can be seen that inthe absence of g(t), the velocity of the particle is given simply by μ₀f(t). Where f(t) is given by Equation (1) there is no net displacementof the particle over a cycle of f(t). Where g(t) is as given above,however, over one cycle, the velocity integrates to yield a distance, d,travelled by the particle of:

$\begin{matrix}{d = {{\int_{t = 0}^{2\;{\pi/\omega}}{\mu_{1}{\sin^{2}\left( {\omega\; t} \right)}\ {\mathbb{d}t}}} = \frac{\mu_{1}\pi}{\omega}}} & (2)\end{matrix}$Thus, the simultaneous application of the two fields imparts a netmotion to the particle. In this example, the net motion is independentof μ₀.

“Particle” is used herein to mean any microscopic or macroscopic thingthat can be moved by scodaphoresis.

The correlation of f(t) and g(t) may be computed according to a suitablecorrelation function such as:

$\begin{matrix}{C_{{f{(t)}},{g{(t)}}} = {\int_{T}^{\;}{{f(t)}{g\left( {t + \lambda} \right)}\ {\mathbb{d}t}}}} & (3)\end{matrix}$where C is the correlation, T is a period of interest, and λ is aconstant time shift. C must have a non-zero value for some value of λ.

Ideally f(t) and g(t) have a large correlation for efficient operationof SCODA, but some SCODA motion can occur even in cases where the chosenfunctions f(t) and g(t) and the chosen value of λ result in small valuesof C. The velocity of the particle undergoing SCODA motion must be afunction of both f(t) and g(t). Further, the velocity of the particle asa result of the application of f(t) and g(t) together must not be thesame as the sum of the velocities resulting from application of f(t) andg(t) independently. That is:{right arrow over (v)}(f(t),g(t))≠{right arrow over (v)}(f(t),0)+{rightarrow over (v)}(0,g(t+λ))  (4)

One set of conditions which is convenient, but not necessary, forscodaphresis is:∫_(−∞) ^(∞) f(t)dt=0,∫_(−∞) ^(∞) g(t)dt=0, ∫_(−∞) ^(∞) v(f(t),0)dt=0,and ∫_(−∞) ^(∞) v(0,g(t))dt=0  (5)where v(f(t),0) is the velocity of a particle as a function of time whenthe particle is interacting only with the driving field f(t); v(0,g(t))is the velocity of a particle as a function of time when the particle isinteracting only with the mobility-varying field g(t); and,∫_(−∞) ^(∞) v(f(t),g(t))dt≠0  (6)in this case, the two fields, acting independently, do not produce anynet motion of the particle. However, the combined effect of the firstand second fields does result in the particle being moved with a netvelocity.

To optimize SCODA one can select functions f(t) and g(t) so that thefirst order velocity of the particles caused by either f(t) or g(t) iszero (so particles have no net drift), and so that the combination off(t) and g(t) acts on the particles to provide a maximum velocity. Onecan select f(t) and g(t) and a phase shift λ to maximize the integral:∫₀ ^(T) {right arrow over (v)}(f(t),g(t+λ))dt  (7)The process in this case runs from time 0 to time T or possibly formultiple periods wherein t rund from 0 to T in each period.

It is not necessary that f(t) and g(t) be represented by sinusoidalfunctions, by the same functions, or even by periodic functions. In someembodiments of the invention, f(t) and g(t) are different functions. Insome embodiments of the invention, f(t) and g(t) are not periodic. FIGS.1A through 1H show some examples of functions f(t) and g(t) that couldbe used in specific embodiments of the invention.

FIG. 1A shows a case wherein f(t) and g(t) are both sine functions withf(t) and g(t) in phase. FIG. 1B shows a case where f(t) and g(t) areboth sine functions with f(t) and g(t) out of phase. As described below,the direction in which particles are caused to move can be reversed byaltering the relative phase of f(t) and g(t).

FIG. 1C shows a case where g(t) is unbalanced. In FIG. 1C, f(t) and g(t)are both triangular functions. In FIG. 1C g(t) has a frequency half ofthat of f(t). In FIG. 1D, f(t) has a square waveform while g(t) has asinusoidal waveform. In FIG. 1E, f(t) and g(t) both have substantiallysquare waveforms. In FIG. 1F, f(t) and g(t) have varying frequencies. InFIG. 1G, f(t) is essentially random noise and g(t) has a value of 1 (inarbitrary units) when f(t) exceeds a threshold 7 and has a value of 0otherwise. In FIG. 1H, g(t) has the form of a series of short-durationimpulses.

As another example, f(t)=sin(ωt), g(t)=1 for

$\frac{2\; n\;\pi}{\omega} < t < \frac{\left( {{2\; n} + 1} \right)\pi}{\omega}$where n is any integer or set of integers (e.g. nε{1, 2, 3, . . . } ornε{2, 4, 6, . . . } or nε{1, 4, 7, . . . }. The integers n do not needto be regularly spaced apart. For example, the methods of the inventioncould be made to work in a case wherein the set of integers n consistsof a non-periodic series. An otherwise periodic waveform f(t) or g(t)could be made aperiodic by randomly omitting troughs (or peaks) of thewaveform, for example.

FIG. 1I illustrates a case where f(t) has a frequency twice that ofg(t). The waveforms of FIG. 1I can produce SCODA motion, for example,where the mobility of particles varies in response to |g(t)|. It can beseen that |g(t)| has larger values for positive-going peaks of f(t) thanfor negative-going peaks of f(t).

While the waveforms shown in most of FIGS. 1A to 1I are symmetrical(i.e. they have the same overall form if inverted in spatial direction)this is not mandatory. f(t) could, in general, be asymmetrical.

Driving Fields

f(t) is referred to herein as a driving function because it drivesmotion of the particles in the medium. In different embodiments of theinvention, f(t) is produced by fields of different types. For example,f(t) may be produced by any of:

-   -   a time-varying electric field;    -   a time-varying magnetic field;    -   a time-varying flow in the medium;    -   a time-varying density gradient of some species in the medium;    -   a time-varying gravitational or acceleration field (which may be        obtained, for example by accelerating a medium containing        particles and periodically changing an orientation of the medium        relative to the direction of the gravitational or acceleration        field);    -   or the like.        In some embodiments, f(t) applies a force to particles that        alternates in direction wherein the magnitude of the force is        the same in each direction. In other embodiments, f(t) combines        a component that alternates in direction and a bias component        that does not alternate in direction such that the magnitude of        the force applied to particles is larger in one direction than        in the other. The bias component may be termed a DC component        while the alternating component may be termed an AC component.

The driving field is selected to interact with the particles ofinterest. For example:

-   -   Where the particles are electrically charged particles (ions for        example), an electric field may be used for the driving field.        Electrically neutral particles may be made responsive to an        electric field by binding charged particles to the electrically        neutral particles. In some cases an electrically neutral        particle, such as a neutral molecule, can be carried by a        charged particle, such as a charged molecule, For example,        neutral proteins that interact with charged micelles may be        driven by an electrical driving field through the interaction        with the driving field and the micelles.    -   Where the particles have dielectric constants different from        that of the medium then an electric field having a time-varying        gradient can drive motion of the particles through the medium by        dielectrophoresis.    -   Where the particles contain magnetic material (for example,        where particles of interest can be caused to bind to small beads        of a type affected by magnetic forces, for example ferromagnetic        beads) a magnetic field may be used for the driving field.    -   Where the particles have magnetic susceptibilities different        from that of the medium then a gradient in a magnetic field may        be used to drive motion of the particles relative to the medium        by magnetophoresis.    -   Where the particles have densities different from that of the        medium then a gravitational or other acceleration acting on the        particles may drive motion of the particles relative to the        medium. An AC acceleration is provided in some embodiments by        exposing the medium to an acoustic field.

The driving field may directly apply a force to the particles or mayindirectly cause motion of the particles. As an example of the latter,the driving field may cause living particles (mobile bacteria forexample) to move in response to their own preference for certainenvironments. For example, some organisms will swim toward light,chemical gradients, or magnetic fields (these phenomena are known aschemotaxis, phototaxis, and magnetotaxis respectively).

Mobility-Varying Fields

The mobility of particles may by altered according to any of a widevariety of mechanisms. For example:

-   -   changing a temperature of the medium;    -   exposing the particles to light or other radiation having an        intensity and/or polarization and/or wavelength that varies in        time with the driving field;    -   applying an electric field to the portion of the medium through        which the particles are passing;    -   applying a magnetic field to the medium through which the        particles are passing (the magnetic field may, for example,        alter an orientation of a magnetic dipole associated with the        particle and thereby affect a coefficient of drag of the        particle or alter a viscosity of the medium which may comprise a        suitable magneto-rheological fluid);    -   applying an acoustic signal to the portion of the medium through        which the particles are passing;    -   causing a cyclic change in concentration of a species in the        medium;    -   exploiting electroosmotic effects;    -   causing cyclic chemical changes in the medium;    -   causing the particles to cyclically bind and unbind to other        particles in or components of the medium;    -   varying a hydrostatic pressure experienced by the medium;    -   varying physical dimensions of the medium to cause a change in        an effective drag experienced by particles in the medium;    -   applying magnetic fields to the medium.        Any effect that varies the mobility of a particle in response to        a driving field, such as an electrophoretic driving field, can        be used.

In some embodiments of the invention, the mobility of particles isvaried by exploiting non-linearities in the relationship between thevelocity of a particle and the intensity of the driving field. Someembodiments apply a second driving field having a component actingperpendicular to the direction of the first driving field but afrequency half that of the first driving field. Applied by itself, sucha second driving field would simply cause particles to oscillate backand forth in a direction perpendicular to the direction of the maindriving field. When applied together with the main driving field,however, such a second driving field can cause particles to have higheraverage speeds relative to the medium for one direction of the maindriving field than for the other direction of the main driving field.This results in a net drift of the particles because of the non-linearrelationship between particle mobility and particle speed. In someembodiments the main driving field has a symmetrical waveform, such as asinusoidal, triangular or square waveform.

A temperature of the medium in which the particles are situated may bealtered in time with the driving field. The changing temperature mayresult in a change in one or more of a conformation of the particles, aviscosity of the medium, a strength of interaction between the particlesand the medium, some combination of these, or the like. The result isthat the mobility of the particles is altered by the change intemperature. The temperature of regions in a medium may be controlled inany suitable manner including:

-   -   directing radiation at the portion of the medium to heat that        portion of the medium;    -   energizing heaters or coolers in thermal contact with the        portion of the medium;    -   causing endothermic or exothermic chemical reactions to occur in        the portion of the medium (or in a location that is in thermal        contact with the portion of the medium); and,    -   the like.        In some embodiments of the invention the medium comprises a        material that absorbs radiation and releases the absorbed        radiation energy as heat. In some embodiment, localized heating        of the medium in the vicinity of the particles being moved is        achieved by irradiating the particles with electromagnetic        radiation having a wavelength that is absorbed by the particles        themselves and released as heat. In such embodiments it can be        advantageous to select a wavelength for the radiation that is        not absorbed or converted to heat significantly by constituents        of the medium so that heating is local to the particles.

Some examples of particles that have mobilities that vary withtemperature are: proteins that can be cyclically denatured or caused tofold in different ways by cyclically changing the temperature; and DNAthat can be cyclically denatured.

Exposing the area of the medium in which the particles are travelling toradiation changes one or more of: a conformation of the particles, aviscosity of the medium, a strength of interaction between the particlesand the medium, some combination of these, or the like. The result isthat the mobility of the particles is altered by changes in theintensity and/or polarization and/or wavelength of the appliedradiation. Some examples of particles that have mobilities that can becaused to change by applying light are molecules such as azobenzene orspiro-pyrans, that can be caused to undergo reversible changes inconformation by applying light. Another example of the use of light tovary the mobilities of particles in a medium is the application of lightto cause partial cross-linking of polymers in a medium containingpolymers.

The intensity of an electric field applied to the medium may be variedin time with the driving field. In some media the mobility of particlesof certain types varies with the applied electric field. In some mediathe particle velocity varies non-linearly with the applied electricfield.

The mobility of particles in a medium may vary with the intensity of anacoustic field applied to the medium. In some cases, an acousticstanding waves in a solution or other medium may cause transientdifferences in local properties of the medium (e.g. electricalresistivity) experienced by particles in the medium thus leading tolocal inhomogeneity in the driving field (e.g. a driving electricfield).

Where mobility of particles is controlled by altering a concentration ofa species, the species having the varying concentration may, forexample, be a species that binds to the particles or a species thataffects binding of the particles to some other species or to a surfaceor other adjacent structure. The species may directly affect a viscosityof the medium.

As an example of the use of electroosmotic effects to control particlemobility, consider the case where the medium in which the particles aremoving is a solution containing one or more polymers. In such solutions,an applied electric field can cause bulk fluid flow. Such a flow couldbe controlled to provide a perturbing stimulus to a pressure or flowinduced driving force, or as a perturbation to an electrical drivingforce, possibly exploiting non-linearities in the onset ofelectroosmotic flow.

Chemical changes that are exploited to control particle mobility may,for example, induce changes in one or more of:

-   -   a conformation of the particles;    -   a conformation of some other species;    -   binding of the particles to one another or to other species or        structures in the medium;    -   binding of species in the medium to one another; viscosity of        the medium; or    -   the like.        The chemical changes may be induced optically, for example, by        optically inducing cross-linking or by optically inducing        oxidation or reduction of photoactive molecules such as        ferrocene. The chemical changes may be induced by introducing        chemical species into the medium. The chemical changes may        include one or more of changes: that alter the pH of the medium;        changes that result in changes in the concentration of one or        more chemical species in the medium; or the like.

Particle mobility may be affected by applied magnetic fields accordingto any of a variety of mechanisms. For example:

-   -   The medium may contain small magnetic beads. The beads may be        linked to polymers in a polymer matrix. By applying a magnetic        field, the beads may be pulled away from a path of the        particles, thereby reducing an effective viscosity of the medium        experienced by the particles.    -   The medium could be a magneto-rheological fluid having a        viscosity that varies with applied magnetic field.

A magnetic field may be used to cause medium viscosity to vary accordingto a two-dimensional pattern. The magnetic field could change in time insuch a manner that the viscosity of the medium varies with position andvaries in time in a manner that provides a synchronous perturbation to aperiodic driving force. As another example, where the particlesthemselves are magnetic, transport and concentration of the particlescould be affected by a magnetic field. The particles could be drivenelectrophoretically. The magnetic field could be switched onperiodically to drive the particles toward a drag-inducing surface, orrelease them from such a surface. The magnetic field could also be usedto make the particles aggregate.

Particles

The methods of the invention may be applied to particles of virtuallyany kind including molecules, ions, and larger particulates. Somenon-limiting examples of particles which may be moved, concentratedand/or extracted through use of the methods of the invention are:

-   -   electrically charged or neutral biomacromolecules such as        proteins, RNA, DNA, and suitable lipids; long polymers;        polypeptides;    -   aggregations of molecules such as micelles or other        supramolecular assemblies;    -   any particles to which magnetic beads or electrically-charged        beads can be attached;    -   living microorganisms; and,    -   the like.

For any particular type of particles, one can attempt to identify asuitable driving field, medium, and mobility-altering field. Since manybiomacromolecules can be electrically charged, it is often suitable touse a time-varying electrical field as the driving field when applyingthe invention to moving and/or concentrating such particles. Further,there are well developed techniques for causing magnetic beads to bondto specific biological materials. Where it is desired to move and/orconcentrate materials which can be caused to bond to magnetic beads thenmagnetic fields may be used as driving fields.

Media

The medium is selected to be a medium through which the particles canmove and also a medium wherein the mobility of the particles can bealtered by applying a suitable mobility-altering field. The medium maycomprise, for example:

-   -   a gel, such as an agarose gel or a performance optimized polymer        (POP) gel (available from Perkin Elmer Corporation);    -   a solution, aqueous or otherwise;    -   entangled liquid solutions of polymers;    -   viscous or dense solutions;    -   solutions of polymers designed to bind specifically to the        molecules (or other particles) whose motion is to be directed;    -   acrylamide, linear poly-acrylamide;    -   micro-fabricated structures such as arrays of posts and the        like, with spacing such that the particles of interest can be        entangled or retarded by frequent collision or interaction with        the micro-fabricated structure;    -   structures designed to interact with molecules by means of        entropic trapping (e.g. Craighead et al., in Science 12 May 2000        Vol. 288);    -   high viscosity fluids such as Pluronic™ F127 (available from        BASF);    -   water; or    -   the like.        The medium is chosen to have characteristics suitable for the        particles being moved. Where the particles are particles of DNA        then suitable polymer gels are the media currently preferred by        the inventors. In some specific embodiments of the invention the        particles comprise DNA and the medium comprises an agarose gel        or a suitable aqueous solution. In some embodiments the aqueous        solution is a bacterial growth medium mixed with a gel such as        an agarose gel.        2D Scodaphoresis

In some embodiments, the particles are constrained to move on atwo-dimensional (2D) surface. In some embodiments the 2D surface isplanar. The 2D surface is not necessarily planar. In some embodiments,the 2D surface comprises a relatively thin layer of a medium, such as agel. In some embodiments the medium is free-standing. The medium may besupported on a substrate. The substrate may comprise a sheet of glass ora suitable plastic such as mylar, for example. In some embodiments the2D layer of medium is sandwiched between the surfaces of two substrates.Where the medium has an exposed surface, the surface may be in air oranother gaseous atmosphere or submerged in a liquid such as a suitablebuffer, an oil, or the like. In some currently preferred embodiments,the medium comprises a layer of a gel sandwiched between two layers ofthicker gel. In an example embodiment, particles move in a layer of a 1%w/v agarose gel sandwiched between two layers of 3% w/v agarose gel.

In some embodiments of the invention, a 2D surface in which particlestravel may be provided by a layer within a medium which has anon-uniform viscosity or a non-uniform concentration of a species thatreduces (or increases) a mobility of the particles. The viscosity orconcentration gradient cause particles to remain in the relatively thinlayer within the medium or on a surface of the medium.

3D Scodaphoresis

SCODA may be used to concentrate particles in three dimensions. This maybe achieved in various ways. In some embodiments, 2D SCODA is performedin a plane. The 2D SCODA may be performed using the electrophoreticSCODA method described below, for example. Z electrodes placed above andbelow the plane could apply an electric field that tends to drive anyparticles that begin to move out of the plane back into the plane.

3D SCODA could also be performed by providing a 6 electrode arrangement,where each electrode is placed on the surface of a body of a medium suchas a gel. Defining X Y and Z axes of such a cube, 2D SCODA would then berun on the 4 electrodes in the XY plane, then the 4 electrodes in the YZplane, then the 4 electrodes in the XZ plane, then repeating in the XYplane and so forth. This would produce a net 3D focussing effect, with anet SCODA force that is radial in three dimensions, but about ⅓ asstrong as the 2D SCODA force for the same electrode voltages.

Control Systems

Any suitable control mechanism may be used to cause a driving field anda mobility-varying field to be applied in a coordinated manner to causeparticles to move by SCODA. In some embodiments of the invention, thetime-variation of the driving field and the mobility-varying field arederived directly from a common source such that their effects on theparticles are correlated. In other embodiments of the invention thedriving and mobility-varying fields are generated under the control of acontroller such as a hard-wired controller, a programmable controller, ageneral purpose computer equipped with suitable interface electronics orthe like. Any suitable control mechanism including those known to thoseskilled in the art of designing scientific equipment may be applied.

EXAMPLES

The following examples illustrate various specific embodiments of theinvention. These embodiments of the invention are considered to beindividually inventive. Some of these examples summarize experimentsthat have been performed and others are prophetic examples.

Example 1 Electrophoretic Concentration of Particles by SCODA

Consider an electrically charged particle that has an electrophoreticmobility, μ in an electric field given by {right arrow over(E)}=cos(ωt)EÊ, where Ê is a unit vector. By definition, the particlewill move with a velocity given by:{right arrow over (v)}=μcos(ωt)E ₀ Ê  (9)From Equation (9), {right arrow over (v)} has a time average of zero. Ifμ varies as a function of time and the Fourier transform of μ has acomponent proportional to cos(ωt) then the time average of v(t) may notbe zero. As a simple example, consider the case where:μ(t)=μ₀+μ₁ cos(ωt)  (10)In this case, the time average of v(t) is:

$\begin{matrix}{\overset{->}{\overset{\_}{v}} = {\frac{1}{2}\mu_{1}E_{0}\hat{E}}} & (11)\end{matrix}$This demonstrates the basic principle that there can be a non zeroelectrophoretic drift even if the time average of the applied electricfield is zero.

Now consider the case where the mobility of a particle is a function ofelectric field strength. While virtually any nonlinearity can beemployed, consider the case where a particle's velocity is parallel tothe direction of a driving electric field and the particle's speed isgiven by:v=kE²  (12)where k is a constant and E is the magnitude of the electric field. Inthis case, the particle's speed is proportional to the square of themagnitude of the electric field. The effective mobility of the particle(i.e. the relationship between small changes in drift velocity, d{rightarrow over (v)}, and small changes in the electric field, d{right arrowover (E)}) varies with the magnitude of the applied electric field.

In Cartesian coordinates:

$\begin{matrix}{{{dv}_{x} = {{\frac{\partial v_{x}}{\partial E_{x}}{dE}_{x}} + {\frac{\partial v_{x}}{\partial E_{y}}{dE}_{y}\mspace{14mu}{and}}}}{{dv}_{y} = {{\frac{\partial v_{y}}{\partial E_{x}}{dE}_{x}} + {\frac{\partial v_{y}}{\partial E_{y}}{dE}_{y}}}}} & (13)\end{matrix}$Where the particle speed varies with the electric field as in Equation(12), Equation (13) reduces to:

$\begin{matrix}{{dv}_{x} = {k\left\lbrack {{\left( {E + \frac{E_{x}^{2}}{E}} \right){dE}_{x}} + {\left( \frac{E_{x}E_{y}}{E} \right){dE}_{y}}} \right\rbrack}} & (14) \\{{dv}_{y} = {k\left\lbrack {{\left( \frac{E_{x}E_{y}}{E} \right){dE}_{x}} + {\left( {E + \frac{E_{y}^{2}}{E}} \right){dE}_{y}}} \right\rbrack}} & (15)\end{matrix}$

To help interpret this, consider the case where E_(y)=0 such thatE_(x)=E. In this case Equations (14) and (15) become:dv_(x)=2kEdE_(x) and dv_(y)=kEdE_(y)  (16)From Equation (16) one can see that the influence on the particlevelocity of perturbations of the electric field has a magnitudeproportional to that of the ambient field. A perturbation having thesame direction as the electric field has twice the influence on theparticle velocity as a perturbation perpendicular to the electric field.

This can be exploited to provide an applied electric field that causesparticles to be concentrated. Consider a plane wherein an appliedelectric field has a constant magnitude, E and the electric fieldrotates in direction at an angular frequency ω so that the components ofthe electric field in x and y directions are given by:E _(x) =E cos(ωt) and E _(y) =E sin(ωt)  (17)Substituting the values from Equation (17) into Equations (14) and (15)yields a result which is the sum of constant terms, sine and cosineterms having an angular frequency ω, and sine and cosine terms having anangular frequency 2ω. A frame of reference can be selected such thatonly the cosine terms having an angular frequency of 2ω contribute tonet particle drift. Evaluating only these terms yields:

$\begin{matrix}{{{dv}_{x} = {{\frac{kE}{2}\left\lbrack {\cos\left( {2\;\omega\; t} \right)} \right\rbrack}{dE}_{x}\mspace{14mu}{and}}}{{dv}_{y} = {{\frac{kE}{2}\left\lbrack {\cos\left( {2\;\omega\; t} \right)} \right\rbrack}{dE}_{y}}}} & (18)\end{matrix}$If a perturbing electric field having the form of a quadrupole fieldthat varies with a frequency 2ω is added to the basic electric fieldspecified by Equation (17) then a net drift of particles can be caused.For a perturbing electric field given by:dE _(x) =−dE _(q) x cos(2ωt) and dE _(y) =dE _(q) y cos(2ωt)  (19)it can be shown that:

$\begin{matrix}{\overset{\_}{d\overset{\rightarrow}{v}} = {\frac{{kEdE}_{q}}{4}\overset{\rightarrow}{r}}} & (20)\end{matrix}$Equation (20) shows that for charged particles at all positions {rightarrow over (r)} there is a time-averaged drift toward the origin with aspeed proportional to k, the coefficient that specifies thefield-dependence of the mobility, E, the strength of the rotating field,and dE_(q), the strength of the perturbing quadrupole field.

The above calculation is for a case where the perturbing quadrupolefield has a magnitude that is small in comparison to the rotating field.This is not necessary in general. FIG. 2 shows the result of a numericalsimulation of the path of a particle in a case where the rotatingelectric field and quadrupole electric field are similar in magnitude.Motion begins at the top right hand side of FIG. 2 and progresses towardthe bottom left over a period of 200 seconds. The applied electricfields are as described in Table I below. Each loop in the spiral pathcorresponds to a cycle of 12 voltage patterns each applied for 1 second.The uniform field amplitude is 3845 V/m at the origin (centre of theelectrode pattern). At the same location, the magnitude of thequadrupole component of the electric field is 4.2×10⁵ V/m² or about 4200V/m at a location 1 mm from the origin.

In many situations it is advantageous to concentrate particles inregions that are free of electrodes. Electrochemical processes atelectrodes can cause damage to DNA and other sensitive materials. Anelectrical field that provides a particle focussing effect, as describedabove, can be provided without the need for electrodes at the locationin which the particles become concentrated.

One can estimate the size of the spot into which particles can beconcentrated from the Einstein-Smoluchowsky equation for diffusion withdrift. A characteristic length scale, R, for the radius of aconcentrated spot is given by:

$\begin{matrix}{R \propto \sqrt{\frac{D}{\mu_{S}}}} & (21)\end{matrix}$where D is the diffusion coefficient for the particles and μ_(s) isgiven by kEE_(q)/4.

FIG. 3 shows apparatus 10 having a simple arrangement that can be usedto practice the invention. A layer 11 of a medium, which may be a gel,such as an agarose gel, is located between four symmetrically arrangedelectrodes 12A, 12B, 12C and 12D (collectively electrodes 12). It hasbeen found to be desirable to provide electrodes 12 in the form of meshelectrodes. A power supply 14 applies individually controllableelectrical potentials V1, V2, V3 and V4 to electrodes 12A through 12Drespectively. Since it is the relative potentials of electrodes 12Athrough 12D that is significant, any one of electrodes 12A to 12D may beheld at a convenient fixed voltage, such as 0 volts, while the voltagesapplied to the other electrodes are varied, if desired.

It is generally desirable to control the potentials applied to theelectrodes to help stabilize the electric stimuli against smallfluctuations due to changing temperature or changing power supplycharacteristics. Separate electrical potential sensing electrodes may beincorporated to provide feedback to a controller representing the actualelectrical potential being applied. FIG. 3A is a schematic view of anapparatus comprising mesh electrodes 12A, 12B, 12C and 12D and separatepotential sensing electrodes 13A, 13B, 13C and 13D (collectivelyelectrodes 13). Large buffer reservoirs 15 maintain an ample supply ofbuffer against evaporation for long runs. Insulating barriers 16separate adjacent reservoirs 15 electrically. Electrodes 13 are locatedin buffer reservoirs 15 and monitor the potential in the buffer.Feedback from electrodes 13 allows a suitably configured controller 14to automatically adjust the voltages on mesh electrodes 12 to compensatefor varying voltage drops across the mesh electrodes/buffer interface.

The magnitude of the applied voltage is chosen to match the size of theapparatus and the particles being separated. For DNA separations inagarose gels electric driving fields of approximately 50V/cm have beenfound to give satisfactory performance. The current supplied will dependupon the electrical conductivity and dimensions of the medium.

The application of the potentials causes electrically charged particlesin medium 11 to move toward a central region 18. FIG. 3 shows groups 17Aand 17B of particles moving toward concentration region 18. As notedabove, the precise waveform according to which the applied electricfields vary is not critical to the operation of the invention. In aprototype embodiment of the invention, the potential variation ofEquations (16) and (18) was approximated by a series of patterns ofdiscrete voltages applied to electrodes 12A through 12D. In theprototype, each cycle was made up of 12 patterns that were each appliedfor 1 second before moving to the next pattern. Table I shows thevoltages applied for each pattern.

TABLE I Voltage Patterns Electrode 12A Electrode 12B Electrode 12CElectrode 12D Pattern (V) (V) (V) (V) 1 0 −66 0 −198 2 132 132 0 0 3 132198 0 198 4 132 198 0 198 5 132 0 0 132 6 0 −198 0 −66 7 0 −198 0 −66 8−132 −132 0 0 9 −132 66 0 66 10 −132 66 0 66 11 −132 0 0 −132 12 0 −66 0−198

In the prototype embodiment of the invention illustrated schematicallyin FIG. 3B, medium 11 was in the form of a gel slab made up of 8-11 mlof 0.25% agarose gel (Agarose 2125 OmniPur available from EMD Chemicalsof Gibstown N.J., USA) forming a 3.8 cm square on an acrylic base in a0.1×Tris-acetate-EDTA buffer. Four electrodes were submerged in the gel.Each electrode extended across one third of one side of the gel boatapproximately 2.5 mm up from the bottom of the gel boat. DNA wasprepared by mixing 8 μl of 500 μg/ml λ phage DNA (48,502 bp, part No.N3011L available from New England Biolabs of Beverly Mass., USA) with 12μl 0.1×TAE. 5 μl spots of the DNA were pipetted directly onto the gelafter the gel had set. A thin covering of TAE was placed on the gel. Thevoltage patterns of Table I were applied to the electrodes. It was foundthat the DNA spots were all carried to a central area of the gel.

FIG. 4 is an example plot of measured DNA velocity as a function ofapplied electric field for the λ DNA used in the prototype embodiment.FIG. 4A is a plot showing time averaged drift velocity (averaged over 15minutes) of the DNA as a function of the radial distance from the originto which the DNA converged. FIG. 4A includes curve 21A which is anumerical estimate of the trajectory of a particle starting at alocation on the X-axis and curve 21B which is a numerical estimate ofthe trajectory of a particle starting at a location X=Y=1.5 cm from theorigin.

FIG. 4B is a plot showing the measured DNA spot distance from the originas a function of time compared to numerical and analytical predictions.The spot position is measured over all spots visible in a given timeinterval. Spot trajectories for spots starting at different radialdistances from the origin are shifted in time so that the start time forspots starting closer to the origin is replaced by the time at whichspots starting farther from the origin reach the starting locations ofthe spots closer to the origin.

In the regime illustrated in FIG. 4B, there was good agreement betweenthe calculated and observed spot trajectories.

For the DNA used in the prototype, D was measured experimentally to be2×10⁻¹² m²/s. μ_(s) was measured to have a value of approximately 1×10⁻³1/s. Using these values, the limiting spot size was calculated to be onthe order of 100 μm. Spot radii on the order of 150 to 250 μm have beenachieved in experiments.

In another experiment, a homogeneous solution of 400 ng/ml λ DNA in 1%agarose gel (0.01×TAE) was subjected to scodaphoresis. The gel wasprepared by mixing 3 ml of 1% agarose gel with 1.5 μl of 500 ng/μl48,502 bp λ DNA and 1.5 μg ethidium bromide (500 ng/ml finalconcentration). The gel was allowed to cool to approximately 65° C. andthen poured into the gel boat. The gel was arranged in a cross shape, asshown in FIG. 3B. Platinum electrodes 19 0.03 mm in diameter werelocated in open electrode regions 20 of the apparatus. The electroderegions were free from gel and filled with 0.01×TAE buffer.

The distance between opposing electrodes was approximately 2.4 cm. Afterapproximately 90 minutes, the λ DNA was found to have been concentratedin a region 21 in the centre of the gel boat in a spot having a fullwidth at half maximum of about 300 μm. The concentration of the λ DNA inthe spot was enhanced by a factor of approximately 3000 to 4000 ascompared to the initial concentration of λ DNA in the gel boat. Theability to cause DNA to be concentrated in an area 21 which is away fromelectrodes is advantageous in various applications.

The concentration factor, F, that can be achieved using a square gelslab having sides of length L is calculated to be approximately:

$\begin{matrix}{F = {\frac{1}{\pi}\left( {{L/200}\mspace{14mu}{µm}} \right)^{2}}} & (22)\end{matrix}$Therefore, other factors being equal, increasing the dimensions of thegel slab can increase the concentration factor. For example,calculations suggest that a 35 cm×35 cm square gel slab could produce aconcentration factor on the order of 10⁶. To achieve the bestconcentration it may be desirable to take steps to inhibit diffusion ofparticles out of the 2D surface in which SCODA is being used toconcentrate the particles.

Electrophoretic SCODA in two dimensions can be performed convenientlyusing four electrodes arranged in two opposing pairs, as describedabove. Other arrangements of three or more electrodes that are notcollinear with one another could also be used. For example SCODA couldbe performed using three electrodes arranged at corners of a triangle.SCODA could also be performed using five or more electrodes arrangedaround a region of a medium.

Since the passage of electrical current through a medium can lead toheating of the medium and most practical media are electricallyconducting to some degree it is desirable to design SCODA apparatus tominimize heating, where practical, and to ameliorate the effects ofheating, where necessary. For example, SCODA may be practised in wayswhich include one or more of:

-   -   cooling the medium through the use of a cooler in physical        contact with the medium, cooling a buffer circulating around the        medium, blowing cool air over the medium or evaporatively        cooling the medium;    -   making the medium very thin, thereby reducing the electrical        current flowing in the medium and improving dissipation of heat        from the medium;    -   placing the medium on a thermally-conductive substrate that acts        as a heat sink;    -   reducing the electrical conductivity of the medium by way of a        chemical treatment or by separating from the medium unneeded        species that give rise to increased electrical conductivity;    -   providing a reservoir of buffer and replenishing buffer        surrounding the medium as the buffer evaporates (see, for        example, FIG. 3A);    -   providing one or more temperature sensors that monitor        temperature of the medium and controlling the temperature of the        medium to remain within an acceptable range by controlling the        electrical current supplied to electrodes; and,    -   using a driving field other than an electrical field.

Example 2 3D SCODA

FIG. 3C shows apparatus similar to that of FIG. 3 that has been modifiedby the provision of additional Z electrodes 22A and 22B. Z electrodes22A and 22B are each maintained at a DC voltage. For negatively chargedparticles, Z electrodes 22A and 22B are kept more negative in potentialthan the 2D SCODA electrodes 12A, 12B, 12C, and 12D. The provision ofthe Z electrodes provides a focussing force in the Z axis, and ade-focussing force in the XY plane of medium 11. The defocussing forceis counteracted by SCODA.

Example 3 3D SCODA

FIG. 3D shows apparatus 24 according to an embodiment of the inventionthat provides 3D concentration of particles in a cube-shaped block ofmedium 11 by alternately performing SCODA using electrodes in XY, XZ,and YZ planes. For example, electrodes 25A, 25B, 25C, and 25D are usedfor concentration in the XY plane. Electrodes 25A, 25E, 25C and anotherelectrode (not visible in FIG. 3D) on the side of medium 11 opposed toelectrode 25E are used for concentration in the YZ plane. Electrodes25B, 25E, 25D and the electrode opposed to electrode 25E are used forconcentration in the XZ plane.

Example 4 Size Selection by SCODA

If desired, SCODA processes can be made to select DNA and similarparticles by size. This may be achieved by suitably adjusting thediffusion coefficient, D (D can be controlled by choice of medium), andthe frequency of the driving field. Using higher driving fieldfrequencies can cause larger particles to be less likely to beconcentrated by SCODA. For example, in one experiment applying a drivingfield having a period of 12 seconds was found to concentrate both long λDNA and shorter DNA fragments from a 1 kB ladder. It was found thatreducing the period of the driving field to approximately 10 ms resultedin concentration of only the shorter DNA fragments but not the longer λDNA fragments. While the inventors do not wish to be bound by anyparticular theory of operation, this size selection may be due to the 10ms period being shorter than the relaxation time for the larger λ DNAfragments and longer than the relaxation time for the shorter DNAfragments.

In the same experiment it was found that SCODA did not concentrateshorter DNA fragments (smaller than a few hundred bp). The selection outof the small sizes may be due to the smaller fragments having highervalues for the diffusion coefficient D.

It is believed that SCODA provides a method for separating supercoiledplasmids from plasmids that are nicked or otherwise degraded.

Example 5 Purification of DNA

Because SCODA can be made selective for different kinds of particles bychoosing a suitable medium and/or combination of driving andmobility-varying fields, SCODA can be used to purify materials, such asDNA. SCODA can be applied to cause DNA (or optionally DNA having aparticular size range) to concentrate at a spot or along a line whileother materials are not concentrated at the spot or line.

For example, in initial experiments, λ DNA was concentrated from amixture of λ DNA and bovine serum albumin (BSA). There was a 10:1concentration ratio of BSA to λ DNA. The λ DNA was concentrated into aspot, as described above. The BSA was not concentrated in the spot.

In some embodiments of the invention, denaturing agents, protease,nuclease inhibitors and/or RNAase are added to a mixture of materialsfrom which the particles are to be separated. Such agents may beprovides to facilitate one or more of:

-   -   reducing the binding of undesired molecules to fragments of DNA        or other molecules that are desired to be concentrated;    -   reducing the amount of RNA present, if so desired;    -   preventing damage to DNA; and/or    -   breaking down the undesired molecules into components that will        not be concentrated by SCODA.

In some cases it may be desirable to use SCODA to separate particles ofinterest from a mixture which includes materials, such as salts, thatcause the medium a high electrical conductivity. For example, bacterialcell cultures are often grown in media having salt contents on the orderof up to 0.4M. In cases where it is desired to use electrophoretic SCODAto separate DNA directly from a cell culture, such as an E. coliculture, the high electrical conductivity will result in higherelectrical currents in the medium. This in turn can lead to heating ofthe medium. This issue may be addressed by one or some combination ofthe heating control techniques discussed above.

Example 6 SCODA with Selective Media

The mobility of a particle in a medium may be made dependent upon thepresence in the particle of a specific DNA sequence by providing amedium with which DNA interacts by binding interactions. For example, agel may be made to include DNA oligonucleotides that are complementaryto the DNA in the particles that it is desired to concentrate. Thecomplementary DNA oligonucleotides may be covalently bonded to the gel.

If the characteristic time required for the particles to bind to thecomplementary DNA oligonucleotides is t_(on) and the characteristic timerequired for the particles to dissociate from the DNA oligonucleotidesis t_(off) then the average drift velocity for a particle in the mediumis given by:

$\begin{matrix}{\overset{\_}{v} = {{\mu(E)}*E\frac{t_{on}}{t_{on} + t_{off}}}} & (23)\end{matrix}$where μ(E) is the field-dependent particle mobility due to reptationeffects. Typically, t_(off) is determined by an Arrhenius relationshipwhile t_(on) is determined by diffusive effects. By selecting particlesto have lengths of 1000 or more nucleotides, reasonable values fort_(off) of 1 second or less can be achieved with practical values ofelectric field (for example, electric fields in the range of 100 to 200V/cm).

FIG. 5 shows estimated particle velocity as a function of electric fieldstrength for three molecules in a sieving matrix comprising covalentlybound oligonucleotides. A first one of the molecules is a perfect matchto the covalently-bound oligonucleotides, a second one of the moleculeshas a single nucleotide mismatch to the covalently-boundoligonucleotides and a third one of the molecules is non-complementaryto the covalently-bound oligonucleotides. DC velocity is shown in solidlines. The SCODA mobility μ_(s) is shown in dashed lines.

It can be seen that there are values for the electric field that resultin the SCODA mobility for particles having DNA that binds to thecovalently bound oligonucleotides being significantly greater than forother particles. At the electric field identified by line 7 the SCODAmobility for particles that perfectly complement the covalently boundoligonucleotides is 25 times greater than it is for non-complementaryand single nucleotide mismatch molecules.

Example 7 Electric Driving Field and Thermal Mobility Varying Field

A demonstration of SCODA was carried out by thermally altering the dragcoefficient of current-carrying solute ions in an electrolyte. Whenapplying an AC potential across an electrolyte solution, andsynchronously raising and lowering the temperature of the solution, anet transport of ions is expected. If the oscillation frequency of theAC potential differs from the frequency of the thermal oscillations, adetectable component of the ionic current should be present at thedifference of the two frequencies, indicating alternating (AC) transportdue to SCODA.

FIG. 6 shows apparatus 30 that may be used to explore electric-thermalscodaphoresis. Apparatus 30 comprises a chamber 32 holding an ionicsolution 33. Electrodes 34A and 34B are immersed in solution 33. Asignal generator 35 applies an electrical signal of a first frequencybetween electrodes 34A and 34B. A heater 36 is in thermal contact withsolution 33. Heater 36 is driven by a power supply 38 so that thetemperature of solution 33 is made to vary at a second frequencydifferent from the first frequency. A detector 40 such as a lock-inamplifier is connected to electrodes 39A and 39B. Detector 40 detects asignal at a frequency equal to the difference of the first and secondfrequencies.

In an experimental prototype apparatus, a microscope slide, cover slipand epoxy were used to construct a chamber holding 300 μL of 2.0M NaClsolution. Two gold wire electrodes were glued to the microscope slide 1cm apart such that they were immersed the NaCl solution. One of theelectrodes was grounded and the other connected through a 1 kΩ resistorto an AC amplifier. Nickel-Chromium Alloy wire (NIC60-015-125-25, Omega,Stamford, Conn.) was glued to one side of the microscope cover slip toallow heating of the solution.

During operation, 1.32 A of current was pulsed to the heater in the formof a 50% duty cycle, square wave at 10 Hz. A small fan runningcontinuously was used to cool the microscope slide during off cycles ofthe heater. A 12 Hz, 3.0V_(RMS) sine wave was applied across theresistor and electrodes. These two signals were mixed and the outputdifference frequency (of 2 Hz) was fed into the reference input of alock-in amplifier (SR830 DSP, Stanford Research Systems, Sunnyvale,Calif.). To measure the periodic current resulting from SCODA, thevoltage across the 1 kΩ resistor was measured with the lock-in amplifierand the 2 Hz component was singled out for analysis. An ionic currentoscillating at 2 Hz was detected.

The driven temperature oscillation of the sample solution was measureddirectly by a thermocouple (0.005-36, Omega) glued to the microscopeslide between the electrodes. The 2 Hz component of the thermocoupleoutput was also analysed using the lock-in amplifier.

We assume the temperature dependent change of the electrolyte'sresistance R₀ is small compared to both the 1 kΩ current-monitoringresistor and the DC resistance of the solution (R_(DC)). The voltageacross the electrodes is:

$\begin{matrix}{{V = {V_{0}{\cos\left( {\omega_{1}t} \right)}}},{V = {4.23\mspace{14mu} V}},{\omega_{1} = \frac{2\;\pi}{T_{1}}},{T_{1} = {\frac{1}{12}\mspace{14mu}\sec}}} & (24)\end{matrix}$The resistance of the salt solution is R=R_(DC)+R₀ cos(ω₂t+φ) where thefrequency of the induced thermal oscillation is ω₂=2π/T₂ with T₂= 1/10s. The total current through the solution is then:

$\begin{matrix}{I_{TOT} = \frac{V_{0}{\cos\left( {\omega_{1}t} \right)}}{R_{D\; C} + {R_{0}{\cos\left( {{\omega_{2}t} + \phi} \right)}}}} & (25)\end{matrix}$Assuming R₀ is small, Equation (24) yields an expression with asinusoidal term at the difference frequency (ω₂−ω₁) whose amplitude isgiven by:

$\begin{matrix}{I = \frac{V_{0}R_{0}}{2\; R_{D\; C}^{2}}} & (26)\end{matrix}$A current having a magnitude of 4 μA at 2 Hz was observed.

Example 8 Electric Driving Field and Optical Mobility Varying Field

In some cases, one can alter the mobility of particles that it isdesired to move by exposing the particles to radiation. In such casesone can practice scodaphoresis by controlling the application ofradiation in time with the driving field such that the average mobilityof the particles is different for the two directions of the drivingfield. For example, one could:

-   -   apply radiation while the driving field is forcing the particles        in one direction and not apply the radiation when the driving        field is forcing the particles in the opposite direction;    -   apply radiation of one wavelength or polarization when the        driving field is forcing the particles in one direction and        apply radiation of a different wavelength or polarization when        the driving field is forcing the particles in the opposite        direction;    -   apply radiation of one intensity while the driving field is        forcing the particles in one direction and apply radiation of a        reduced intensity when the driving field is forcing the        particles in the opposite direction;    -   apply radiation having a time-varying intensity g(t) that has a        non-zero correlation with the driving field;    -   and so on.

In some cases it is not practical or desirable to use radiation to alterthe mobility of the particles themselves but it is practical to bind tothe particles other molecules that have mobilities that can becontrolled by applying radiation. The other molecules may, for example,have conformations that can be changed by applying radiation or may bindto the medium in a manner that can be controlled by applying radiation.

In some embodiments, azo-benzene is attached to particles to besubjected to scodaphoresis. Azo-benzene can isomerize from the trans- tocis-form upon exposure to UV light (300-400 nm). The azo-benzene revertsto its trans-form when it is exposed to light having a wavelengthgreater than 400 nm. In some embodiments, spiro-pyrans is attached tothe particles. Exposure of the ‘closed’ form of spiro-pyrans to UV lightinduces isomerization to yield an ‘open’ coloured merocyanine species.The spiro-pyrans reverts to its ‘closed’ form on exposure to visibleradiation. The transition between these two forms is accompanied bychanges in the polar nature of the molecule.

Where radiation is used to vary the mobility of particles, differentradiation fields may be applied in different areas to achieveconcentration of the particles. For example, consider the apparatus 41shown in FIG. 7. In apparatus 41 a medium 11 is located between twoelectrodes 42A and 42B. An AC power supply 43 applies an AC electricalsignal 44 (FIG. 7A) between electrodes 42A and 42B.

Light projectors 46A and 46B respectively illuminate portions 45A and45B of medium 11. A control 47 causes light projector 46A to illuminatearea 45A only when signal 44 creates an electrical field in a firstdirection. Control 47 causes light projector 46B to illuminate area 45Bonly when signal 44 creates an electrical field in a second directionopposed to the first direction. The result is that particles in medium11 converge on line 49 at the boundary of areas 45A and 45B from bothsides.

Many alternative constructions can be used to illuminate areas 45A and45B in time with a driving field. For example:

-   -   Light from a single lamp could be steered by a suitable optical        system to illuminate areas 45A and 45B in alternation;    -   Light from one or more lamps could be blocked from areas 45A and        45B in alternation by a suitable arrangement of mechanical or        electromechanical filters, shutters, masks or other devices        having a controllable light transmission or reflection; and,    -   so on.        Focussing in the Y direction may be achieved by rotating the        light pattern and electrical field by 90 degrees relative to        medium 11.

Electrical/optical SCODA may be used to cause particles to congregate atan array of spots or along a number of lines. This can be achieved byapplying a patterned light field to the area of medium 11. This may beused to provide samples of DNA that are concentrated along spots orlines for example. Various biological applications require an array ofspots or lines of DNA.

FIGS. 8A through 8D shows a possible arrangement of four masks 50Athrough 50D that can be used to concentrate particles into an array of16 spots. Masks 50A and 50B are complementary to one another. Masks 50Cand 50D are complementary to one another. Mask 50A is applied while adriving field causes particles to move in a direction 51A. Mask 50B isapplied when the driving field causes particles to move in direction51B. It can be seen that particles will be concentrated along the fourlines 52A, 52B, 52C, and 52D if the driving field is alternated betweendirections 51A and 51B while masks 50A and 50B are applied as describedabove. Similarly, by alternately applying mask 50C with the drivingfield in direction 51C and mask 50D with the driving field in directionMD, particles will be concentrated along the four lines 53A, 53B, 53C,and 53D.

Eventually, after a number of cycles, particles will be concentrated inspots 54 at the intersections of lines 52A to 52D and lines 53A to 53Das shown in FIG. 8E. The particles may comprise, for example, desiredDNA or other molecules having attached azobenzene groups. The order inwhich masks 50A to 50D are applied (together with their correspondingdriving fields) can be varied. In simple embodiments, concentration inthe X direction is performed first using masks 50A and 50B and thenconcentration is performed in the Y direction using masks 50C and 50D.

Example 9 Optical Mobility Variation by Localized Viscosity Change

Particles to be concentrated by SCODA are located in a medium having aviscosity that varies with temperature. The mobility of the particles isdependent on the viscosity of the medium. The particles have anabsorption band. Upon absorbing radiant energy having a wavelength inthe absorption band, the particles release the absorbed energy as heat.

An alternating driving field of any suitable type is applied to theparticles. The particles are illuminated with radiation having awavelength in the absorption band and an intensity g(t). g(t) isselected so that g(t) has a non-zero correlation with the force f(t)applied to the particle by the driving field. When g(t) has a largevalue, the rate at which each particle releases thermal energy is largerthan it is when g(t) has a smaller value. The thermal energy released bythe particles in response to the absorbed radiation heats thesurrounding media and locally alters its viscosity and thus the particlemobility.

Example 10 Fluid Flow as Driving Field

A SCODA driving field may be created by causing the medium in which theparticles are situated to have a velocity that alternates in direction.For example, the medium may comprise a fluid in a pipe or capillary tubethat is caused to flow back and forth in the pipe. The mobility of theparticles may then be varied, either by causing the particles tointeract with an externally applied field or by causing the particles tointeract with a wall of the pipe in which they are located.

For example, consider a back and forth flow of a liquid in a pipe, inwhich molecules are suspended whose size is comparable to the pipediameter (e.g. large DNA in a micron size capillary). Now, vary thecapillary diameter (e.g. by providing the capillary with flexible wallssuch as walls of a silicone material and subjecting the capillary toexternal pressures) such that when the flow is in one direction, themolecules interact more frequently with the capillary wall and areretarded.

Example 11 Use of Cyclic Dilution/Concentration to Vary Mobility

Cyclic dilution/concentration may be used to vary the mobility ofparticles, especially where the particles are travelling along at ornear a surface. The concentration or viscosity of the medium in whichthe particles are travelling may be modulated over time to correlatewith the electrical or other field driving motion of the particles.

FIG. 9 shows apparatus 60 in which particles are travelling along afluid layer 62. The particles are driven by an alternating electricfield applied between electrodes 64A and 64B. A sprayer 66 dilutes fluidlayer 62 by applying a solvent when the electric field is in a firstdirection. A vacuum valve 68 is opened to cause solvent from fluid layer62 to evaporate, thereby increasing the concentration of fluid layer 62when the electric field is in a second direction. Valve 68 and sprayer66 are operated by a suitable control system (not shown).

Example 12 Pathogen Detection

In environmental sampling it is sometimes necessary to determine whethercertain pathogens are present within relatively large volumes (e.g. 1 Lor 10 L) of fluid (or solid material that can be dissolved in a fluid).Such volumes are too large for PCR to be performed in a cost effectivemanner in most cases. Filters can be used to concentrate DNA but suchfilters tend to clog. SCODA may be used to concentrate such pathogens,if present, in a sample. The sample can first be coarsely purified, thenintroduced into a medium in which particles of interest in the samplecan be concentrated by scodaphoresis. For example, the particles may beintroduced into a gel by mixing the sample with buffer and gel materialto form a large volume gel. The buffer may include detergents or otheragents to help lyse the pathogens and release their DNA into solution.

2D or 3D SCODA can them be performed to concentrate all or most of theDNA in the volume at a central location. The DNA at the central locationis contained in a volume of gel that is manageable by normal means. Theconcentrated DNA may then be PCR amplified to detect specific pathogens.DNA can optionally be extracted from the gel using, for example, acommercial kit (e.g. Qiagen™) or using the I-ZIFE extraction methodsdescribed below before performing PCR amplification. In the alternative,a piece of the gel including the concentrated DNA may be subjected toPCR. The gel tends to melt during the PCR reaction does notsignificantly adversely affect the PCR amplification in someapplications.

Example 13 Magnetic Control of Particle Mobility

FIG. 10A shows a medium 60 comprising a polymer matrix. The mediumincludes polymers 62 linked to magnetic beads 64. The magnetic beadscould be of the type currently available and used for DNA extractions. Amagnetic field generated by a suitable magnet 66 could be turned on topull magnetic beads 64 and the associated polymers 62 to one side of themedium as shown in FIG. 10B. the result is that a region 68 of themedium becomes less viscous. The magnetic particles could be released byswitching off the magnetic field to resume the situation illustrated inFIG. 10A wherein the medium in region 68 is more viscous than it is withthe magnetic field on.

The magnetic field may be patterned in two dimensions and changed overtime such that the viscosity of the medium is a function of both timeand position in the medium.

In an alternative embodiment illustrated in FIGS. 10C and 10D, theparticles being transported are themselves magnetic. The driving field,may, for example, be an electrical field. A magnetic field could beswitched on periodically to drive the particles toward a drag-inducingsurface 67. The magnetic field could be switched off to release theparticles from surface 67.

In other embodiments, the medium comprises a magneto-rheological fluidso that the medium has a viscosity that inherently varies with theapplied magnetic field.

Example 14 Acceleration as a Driving Field

A gravitationally induced flow in a density gradient may be used as adriving field. consider, for example, a tube filled with a medium, suchas a solution in which heavier or lighter particles are suspended. Thetube is located in a centrifuge so that the particles tend to traveltoward one end of the tube. The orientation of the tube is periodicallyreversed. A suitable mobility-varying field could be applied in timewith the reversals of orientation so that the particles are caused toachieve net motion in one direction along the tube.

Example 15 SCODA for Desalination

Consider an electrically insulating capillary filled with a salinesolution. If the fluid in the capillary is caused to flow then aparabolic velocity profile is established in the capillary. Fluid flowsmore quickly at the centre of the capillary than near the capillarywalls. If an electric field is established across the capillary, ionswill build up preferentially within a Debye length (charge screeninglength) of the capillary walls as required to cancel the appliedelectric field. This changes the radial distribution of ions in thecapillary and thus changes the average velocity of the ions. If thefluid flow is caused to reverse periodically and the electric field isapplied only for one direction of flow then there will be a nettransport of ions in one direction along the capillary until the SCODAinduced drift is counteracted by diffusion from the accumulated iondensity gradient along the capillary.

By applying a slight DC bias to the AC fluid flow in a directionopposite to the direction of ion transport, the fluid emerging from thecapillary will have a reduced ion content.

Some Possible Variations to SCODA Methods and Apparatus

As described in Example 6 above, where the driving field and mobilityvarying field are not synchronized with one another, the result is thatthere is a flow of particles back and forth between two locations as therelative phases of the driving and mobility-varying fields vary. Thisability to move particles back and forth between two locations at acontrollable frequency may be useful in various contexts.

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof. For example:

-   -   It is not necessary to generate one of the driving and        mobility-varying fields. A suitable existing field, which could        comprise a field already present for some other purpose or even        noise could be used for one of f(t) or g(t). This existing field        can be detected and a second field may be applied in time with        the detected field so that the mobility of particles is altered        in time with a driving field to produce a net drift.    -   Physically rotating electrodes at constant voltage could be used        to simulate the rotating field used for 2D SCODA.    -   Small DC biases can be used to shift the position of focused        spots.    -   Some embodiments of the invention provide wells in the medium at        locations where particles are expected to be concentrated by        SCODA. The wells may be filled with a suitable buffer solution.        Particles can diffuse into the wells as a result of SCODA        induced concentration gradient. Particles can be extracted from        the wells with a pipette or other transfer device.        Particle Extraction Methods and Apparatus

Various methods for moving and/or concentrating particles are describedabove. Often, after particles have been concentrated, it is desirable toremove the particles from the medium in which they have beenconcentrated. For example, where DNA is concentrated in a gel, it isoften desirable to extract the DNA from the gel for subsequentprocessing.

The following description explains methods and apparatus which may beused to extract particles from a medium. These methods and apparatus maybe called “interface zero integrated field electrophoresis” (“IZIFE”)methods and apparatus. IZIFE may be used to extract particles that havebeen moved to a location and/or concentrated by a SCODA method IZIFEalso has more general application in extracting particles from media.

IZIFE exploits differences in the mobility properties of particles indifferent media (such as a gel medium and a buffer medium). Some chargedparticles (such as molecules of DNA) exhibit an electrophoretic mobilityin gel solution (such as agarose gel) that depends on the magnitude ofthe electric field applied. Such particles can be caused to drift in onedirection in such media by applying an electric field that variesasymmetrically with time. However, when those particles are in buffer orfree solution, they have an electrophoretic mobility which is constantor at least has a much lower dependence on electric field strength.Therefore, the particles stop drifting if they are carried into a mediumwhere they have a mobility that does not vary with applied field.

Application of ZIFE (zero-integrated-field-electrophoresis) to a gelcontaining charged particles will cause the particles to drift in thedirection that yields the greater mobility. If the particles enter aregion containing a buffer or free solution, they will stop drifting.Continued application of a zero time-averaged electric field causes nonet drift on the particles in the buffer solution. Therefore, theparticles tend to become concentrated in the buffer solution adjacent tothe interface between the buffer solution and the gel.

The extraction methods detailed herein permit particles to be extractedfrom a medium. The invention may be applied to extracting chargedbiopolymers such as DNA, RNA and polypeptides from electrophoresismedia, for example. Some embodiments of the invention use ZIFE to moveparticles from a medium, such as an electrophoresis gel, into anadjacent fluid. ZIFE is a form of Alternating Current (AC)electrophoresis where, the polarity of an applied electric fieldreverses periodically and the time-averaged electric field is zero. Theintensity of the electric field is greater in one polarity than in theother.

FIG. 11 is a graphical illustration of an exemplary electric field pulseused in ZIFE. As shown in FIG. 11, the pulse comprises an electric fieldE₁ applied in the positive, or “forward”, direction for a time t₁,followed by an electric field E₂ applied in the negative, or “reverse”,direction for a time t₂. If E₂=−E₁/r_(ε) (where r_(ε) is the fieldratio), and t₂=t₁r_(ε), then the time-averaged electric field is zero.The time-averaged electric field is graphically represented by theshaded areas in FIG. 1. The “positive” shaded areas (corresponding toE₁) cancel the “negative” shaded areas (corresponding to E₂). Overallthere is a zero net electric field. If the time-averaged electric fieldis exactly zero, then the ZIFE process is unbiased. If the time-averagedelectric field deviates from zero, then the ZIFE process is biased

The velocity ν of a particle moving in a local electric field ofamplitude E and having an electrophoretic mobility μ is given by:ν=μE  (27)For linear systems, μ is constant. Particles having constantelectrophoretic mobility have no net migration in a medium (i.e. theirnet velocity is zero) when ZIFE is applied to the medium. By contrast,in non-linear systems, particles have an electrophoretic mobility thatis dependent on electric field amplitude. In such non-linear systems,there is a net migration of the particles in the direction that yieldsthe greater mobility. In such a non-linear system, the particle velocitymay be given by:ν=μ(E)E  (28)

Suppose that charged particles in a medium have a field-dependentelectrophoretic mobility of the form:μ(E)=μ₀ +kE  (29)It can be seen that the mobility of these particles increases with theamplitude of the electric field E. The distance d traveled by theparticles under the influence of a constant electric field E is given byd=νt. If an electric field pulse of the form shown in FIG. 11 isapplied, the particles will travel a greater distance during t₁ (whilethe pulse has the greater field amplitude) than the distance traveledduring t₂. This can be shown by applying Equations (27) and (29) to thedistance traveled by the particles. Hence, there is a net drift ofparticles in the “forward” direction, i.e. the direction in which theelectric field of amplitude E₁ is applied.

This net drift behavior has been demonstrated by DNA molecules inagarose gels. In such gels, DNA molecules have an electrophoreticmobility of the form given by Equation (28). The field dependence ofmobility arises from interactions between the DNA molecules and the gel.Therefore, ZIFE can be applied to DNA in an agarose gel to directparticles made up of DNA in a desired direction.

By contrast, application of ZIFE to DNA molecules in a buffer or freesolution does not produce a net migration of DNA. This is because themobility of DNA molecules in buffer solution is not field dependent. Thedifferences in mobility properties of DNA in two media (e.g. a bufferand a gel) can be exploited to move particles from within one mediuminto another medium where the particles can be accumulated. This can bedone by applying a ZIFE field across an interface between the two media.

Consider, for example, applying a ZIFE field across an interface betweena gel in which there are DNA molecules and a buffer solution. ApplyingZIFE to the molecules of DNA in the gel causes the molecules to migratein the gel toward the gel-buffer interface. Once those molecules enterthe buffer, the molecules will stop migrating. The ZIFE field may have asmall bias in the direction which tends to move the molecules from thebuffer toward the gel. This bias tends to prevent the molecules fromdiffusing too far away from the interface after they enter the buffer.The bias may prevent the molecules from encountering the electrode usedto create the ZIFE field. The bias is small enough that the particles inthe gel continue to move toward the interface (i.e. the ZIFE velocity isnot overcome by the net drift resulting from the bias).

Apparatus according to one embodiment of the invention is shown in FIGS.12A and 12B. FIG. 12A shows molecules of DNA in a gel, prior toextraction, and FIG. 12B shows molecules of DNA concentrated in abuffer, after extraction from the gel. An extraction apparatus 200comprises a gel boat 120 (which may be shaped as a rectangular box)containing a gel 122, such as agarose gel. Gel 122 fills a substantialvolume of gel boat 120. Preferably gel 122 is separated from each ofelectrodes 130B by a buffer solution in a reservoir 124. Reservoirs 124are separated from one another so that the buffer does not provide shortcircuit paths between electrodes 130B.

As shown in FIG. 12A, prior to extraction, molecules of DNA 128A areconcentrated in a column in gel 122. Molecules 128A are typically notconcentrated in such form when left in their natural state. Prior tobeing concentrated, molecules 128A are typically distributed throughoutgel 122. Molecules 128A may be concentrated into a column as shown inFIG. 12A through the use of SCODA, as described above. In thealternative, molecules 128A may be concentrated by another method. Forexample, the molecules to be extracted may be the molecules of a band ofDNA separated by conventional DC electrophoresis or PFGE

Concentration of molecules 128A in a region of gel 122 is not requiredprior to extraction. However, concentration is preferable to facilitatemore efficient extraction of the molecules.

A capillary 125 containing a small amount of buffer solution is insertedinto gel 122 so as to surround the molecules 128A to be extracted.Capillary 125 may be inserted by a robotic device which permits thelocation of insertion to be carefully controlled and which inserts thecapillary with minimal disturbance of the gel. The robotic device maycomprise a multi-axis positioner, such as an X-Y positioner, thatpositions capillary 125 over a desired location in a medium and thenlongitudinally extends the capillary into the medium. After capillary125 is inserted into gel 122, the top portion of capillary 125 containsbuffer solution, while the bottom portion of capillary 125 contains gel122. The buffer solution in capillary 125 provides an extractionreservoir 126 adjacent to gel 122. Extraction reservoir 126 meets gel122 at a buffer-gel boundary 121. The arrangement of buffer and gel incapillary 125 forms a buffer-gel interface 131. A pipette 129 isprovided above capillary 125 to suction molecules 128A after they havemigrated into extraction reservoir 126.

To provide the electric fields required for electrophoresis, anelectrode 130A is located near the tip of pipette 129. Electrode 130A ispreferably located sufficiently far from the interface that theextracted molecules do not encounter electrode 130A while the ZIFE fieldis being applied. A plurality of electrodes 130B are located in bufferreservoir 124. The electrodes may be made of platinum, for example. Moreelectrodes may be provided than those shown in FIGS. 12A and 12B.

The tip of pipette 129 is filled with a small amount of buffer so as toprovide conductivity between electrodes 130A and 130B when the pipetteis inserted in capillary 125. In one embodiment, electrodes 130B areganged to a fixed common potential (for example, electrodes 130B may begrounded), while electrode 130A is set to a different potential. Avarying electric field can be applied across buffer-gel interface 131 byvarying the potential of electrode 130A.

To perform Interface-ZIFE, a zero time-averaged pulsed electric field isapplied across buffer-gel interface 131. The pulsed electric field maybe of the form shown in FIG. 1, for example. To cause molecules 128A tomigrate in the desired direction (i.e. toward extraction reservoir 126),an electric field having an amplitude E₁ is applied in the directiontoward extraction reservoir 126, while an electric field having anamplitude E₂ is applied in the opposite direction. E₁ and E₂ are chosensuch that the particles to be extracted have a greater mobility underthe influence of E₁ than they do under the influence of E₂. For typicalmolecules and media E₁>E₂. The polarity is selected so that theparticles are driven toward interface 131 under the influence of E₁.

Application of Interface-ZIFE across buffer-gel interface 131 will causemolecules 128A in gel 122 to drift toward extraction reservoir 126.After some time, some of the molecules 128A will cross buffer-gelboundary 121 and enter into the buffer in extraction reservoir 126. Oncethese molecules reach extraction reservoir 126, Interface-ZIFE has nonet drift effect on the molecules and the molecules thus stop drifting.Eventually all (or most) of molecules 128A will cross the buffer-gelboundary 121 and migrate into extraction reservoir 126. Molecules 128Abecome concentrated in the buffer adjacent the interface.

FIG. 12B shows molecules 128B (corresponding to molecules 128A in FIG.2a ) that have migrated from gel 122 into extraction reservoir 126.Thus, Interface-ZIFE can be used to collect and concentrate molecules128B in extraction reservoir 126. Pipette 129 or another device can thensuction molecules 128B from extraction reservoir 126, thereby completingthe extraction process.

FIG. 13 shows a glass capillary in an extraction experiment in which DNAmixed with a liquid gel was allowed to set within a capillary tube.Buffer was added to an upper portion of the capillary. The techniquesdescribed above were used to extract the DNA. An image of the capillarywas captured at various times (0 minutes, 60 minutes, 120 minutes) toshow the effects of Interface-ZIFE applied to a buffer-gel interface.The buffer is a TAE (Tris-Acetate-EDTA) buffer and the gel is an agarosegel containing DNA. To perform this experiment, 100 μL of liquid 1%agarose gel, mixed with 5 μL λ DNA and 2.5 μg EtBr, was pipetted intothe lower portion of a 2.5 mm inner diameter glass capillary and allowedto solidify. The upper portion of the capillary was filled withapproximately 50 μL of 0.1×TAE buffer and a first platinum electrode wasinserted into the buffer. The bottom of the capillary was then submergedin a shallow reservoir of 0.1×TAE buffer with a second platinumelectrode.

Interface-ZIFE was performed with these conditions: periodically, avoltage V₁=200 V was applied to the first electrode for a time t₁=8 s,then a voltage V₂=−100 V was applied to the second electrode for t₂=16s. The electric field was pulsed for 2 hours. The electrodes wereseparated by 5 cm. Over the course of the experiment, the upper half ofthe capillary remained filled with buffer and the DNA remained in arelatively small volume (approximately 20 μL). As shown by the images ofthe capillary, there is a progressive migration of DNA through a gel andconcentration of the DNA in a small amount of buffer above the gel.

If extraction reservoir 126 is sufficiently small, then molecules 128Bthat are concentrated in a region in extraction reservoir 126 will leavetheir concentrated region only by diffusion, which is slow over longdistances. Convective mixing of molecules 128B and extraction buffer 126should be minimized to maintain molecules 128B in their concentratedregion. To minimize convective mixing, capillary 125 should preferablyhave a small diameter. Moreover, extraction buffer 126 and gel 122 arepreferably kept at the same temperature.

In one embodiment, pipette 129 comprises a mechanized pipettor withbuilt-in electrode 130A. The mechanized pipettor aspirates buffer into adisposable pipette tip, then partially dispenses the buffer to cover thegel inside capillary 125 so that there is conductivity betweenelectrodes 130A and 130B. Computer monitoring may be used to monitor thecurrent between electrodes 130A and 130B during extraction, and detectsuch problems as bubbles or evaporation that may create an open circuitbetween the electrodes. After extraction is complete, the remainingbuffer in the pipette tip is disposed of, and the pipette tip may returnto capillary 25 to extract further samples of particles. Mechanizedpipetting may reduce unnecessary pipette tip motion so that there isminimal mixing of the concentrated particles with the surroundingbuffer. This minimizes the extraction volume and hence increases finalconcentration of the particles to be extracted.

In another embodiment, instead of inserting a capillary filled withbuffer into the gel, the gel may be cast with a cavity. The cavity isfilled with a buffer solution, and a pipette having an electrode isinserted into the buffer. The cavity functions similarly to thecapillary in collecting the particles for extraction. Molecules may becaused to enter such a cavity from the surrounding medium by generatinga concentration gradient between the medium and the cavity by SCODA.

Interface-ZIFE extraction of DNA mixtures from gels may be applied toselectively extract DNA fragments according to their size. If cycletimes t₁ and t₂ for the electric field pulse are chosen to besufficiently small, the relaxation or re-orientation time of the DNAmolecules becomes significant and introduces a length-dependence in themigration velocity of the molecules. FIG. 14 is a graph illustrating theDNA fragment velocity during an experiment as a function of fragmentlength and cycle times t₁ and t₂. In that experiment, DNA fragments ofdifferent lengths were linearly separated using standard DCelectrophoresis in a 1% agarose gel (0.1×TAE). ZIFE was then applied (ina direction perpendicular to that in which the DC electrophoresis wasperformed) to observe non-linear velocity of the fragments.

FIG. 15 shows a comparison between a DNA fragment mix and the fragmentdistribution of the same mix, after Interface-ZIFE extraction. The mixcomprised 2 μL λ DNA (48 kb, 500 ng/μL) and 4 μL 1 kb DNA ladder (0.5-10kb, 500 ng/μL) and was run in 100 μL of 1% agarose gel applying theInterface-ZIFE extraction method described above. A pulsed electricfield was applied, generated by a voltage V₁=200V applied to theelectrode in the pipette for a time of t₁=25 ms, which alternated with avoltage V₂=−100V applied to the electrodes in the gel for a time oft₂=50 ms. The pulsed electric field was applied for 3 hours. Thisprocess extracted DNA into 0.1×TAE buffer which was mixed with loadingdye and inserted into the well of a 1% agarose gel, along with a controlfrom the original mix, for standard DC electrophoresis. The X DNA bandand short (less than 1 kb) fragments were not extracted from the gel.The size selection of Interface-ZIFE may be applied to longer fragments(100-200 kb) as well.

Parameters that can be varied to optimize extraction speed, extractionefficiency and DNA fragment length selectivity, include: magnitude ofthe electric pulsed field; frequency (cycle times) of the electricpulsed field; composition of the buffer in extraction reservoir 126;composition of gel 122; operating temperature; and the degree ofconcentration of molecules 128A.

The methods and apparatus disclosed herein may be applied for extractingcharged particles from a medium where the particles are concentrated ina particular region of the medium (such as DNA molecules concentrated ina column or pillar in gel). However, the methods and apparatus are notlimited to such application. They may also be employed to extractcharged particles that are uniformly dispersed in the medium, located orconcentrated in particular regions or bands, or otherwise distributed inthe medium. Using the methods and apparatus disclosed herein, chargedparticles, and in particular biopolymers (for example, DNA, RNA andpolypeptides), may be extracted from acrylamide, linear poly-acrylamide,POP (Perkin Elmer), agarose gels, entangled liquid solutions ofpolymers, viscous or dense solutions, solutions of polymers designed tobind specifically to the molecules whose motion is being directed,simple aqueous solutions, and the like. Interface-ZIFE used inconjunction with SCODA-based electrophoresis (for concentrating the DNAin a region) can be used to extract bacterial artificial chromosomes,plasmids and high molecular weight or genomic DNA.

IZIFE can be used to extract only selected particles from a medium.Particles having velocities that depend only linearly on the magnitudeof an applied driving field will simply oscillate back and forth whenexposed to an IZIFE driving field. Such particles will therefore remainin the medium while other particles having velocities having anon-linear dependence on applied field can be extracted from the medium.In some cases the IZIFE driving field can be constructed so thatdifferent particle species drift toward the second medium at differentrates. The concentration of the different species at the interfacebetween the media will therefore vary over time. A species which has ahigh net drift velocity under IZIFE will be extracted from the firstmedium before a species which has a lower net drift velocity.

By terminating IZIFE before slower species have been extracted from thefirst medium, the relative concentration of species having faster netdrift velocities can be increased. By removing faster species that haveaccumulated at the interface before slower drifting species have arrivedat the interface, one can increase the relative concentration at theinterface of species having slower net drift velocities under IZIFE

Some Possible Variant Particle Extraction Methods and Apparatus

As will be apparent to those skilled in the art in the light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof, including but not limited to the following:

-   -   The extraction of uncharged, or electrically neutral, molecules        may be accomplished using the methods and apparatus disclosed        herein if those molecules are carried by charged molecules. For        example, neutral proteins that interact with charged micelles        may be extracted electrophoretically through their interaction        with the micelles.    -   The waveform used for implementing ZIFE may be biased in one        direction or the other. Biased ZIFE may facilitate selective        separation of the particles according to their size.

Instead of using IZIFE to extract particles from a first medium into asecond medium, one could use SCODA to extract particles from the firstmedium into a second medium. In some such embodiments a SCODA drivingfield that alternates in direction is directed across an interfacebetween the first and second media. The SCODA driving field may, forexample, be directed substantially perpendicularly to the interface. ASCODA mobility-varying field may be selected such that themobility-varying field affects the mobility of the particles in thefirst medium so as to cause the particles in the first medium to travelin a direction toward the second medium. The mobility-varying field isselected to affect the mobility of the particles the second medium to adegree substantially less than it affects the mobility of the particlesin the first medium. In the best case the mobility-varying field doesnot affect the mobility of particles in the second medium. In thisexample, the SCODA effect causes particles to be transported from thefirst medium into the second medium where the particles becomeconcentrated at the interface between the first and second media. In analternative embodiment, the mobility-varying field is applied only tothose particles that are within the first medium so that the particlesdrift by SCODA into the second medium and become concentrated in thesecond medium.

Where a component (e.g. a power supply, electrode, assembly, device,circuit, etc.) is referred to above, unless otherwise indicated,reference to that component (including a reference to a “means”) shouldbe interpreted as including as equivalents of that component anycomponent which performs the function of the described component (i.e.,that is functionally equivalent), including components which are notstructurally equivalent to the disclosed structure which performs thefunction in the illustrated exemplary embodiments of the invention.

Accordingly, the scope of the invention is to be construed in accordancewith the substance defined by the following claims.

The invention claimed is:
 1. A method for causing motion of targetnucleic acids in contact with a separation medium, the methodcomprising: applying a first time-varying driving field to the targetnucleic acids with a first electrode adjacent the separation medium;applying a second time-varying driving field to the target nucleic acidswith a second electrode adjacent the separation medium; applying a thirdtime-varying driving field to the target nucleic acids with a thirdelectrode adjacent the separation medium; and synchronously varying themobility of the target nucleic acids in the medium, wherein the first,second, and third electrodes are adjacent a perimeter of the separationmedium and approximately equidistant from each other.
 2. The method ofclaim 1, wherein the mobility of the target nucleic acids is varied bychanging a temperature of the separation medium.
 3. The method of claim2, wherein the temperature is varied with Joule heating.
 4. The methodof claim 1, wherein the separation medium comprises a polymer.
 5. Themethod of claim 1, wherein the separation medium comprises boundoligonucleotides.
 6. The method of claim 5, wherein the boundoligonucleotides are complimentary to a portion of the target nucleicacids.
 7. The method of claim 1, wherein the target nucleic acidscomprise 200 or fewer nucleotides.
 8. A method of separating a targetnucleic acid from a sample comprising other nucleic acids, comprising:loading a sample comprising a target nucleic acid and other nucleicacids on a separation medium; and applying a time-varying driving fieldto the medium while synchronously varying the mobility of the nucleicacids in the separation medium, thereby separating the target nucleicacid from the other nucleic acids by causing the target nucleic acid tobind and unbind to the medium at a sequence-specific rate or asequence-specific strength that is different from the rate or strengthof the other nucleic acids having different sequences.
 9. The method ofclaim 8, wherein the mobility of the nucleic acids in the separationmedium is varied by changing a temperature of the separation medium. 10.The method of claim 9, wherein the temperature is varied with Jouleheating.
 11. The method of claim 8, wherein the separation mediumcomprises a polymer.
 12. The method of claim 8, wherein the separationmedium comprises bound oligonucleotides.
 13. The method of claim 8,wherein the target nucleic acid comprises 200 or fewer nucleotides.